Stroke causes CNS injury associated with strong fast microglial activation as part of the inflammatory response. In rat models of stroke, sulphonylurea receptor blockade with glibenclamide reduced ...cerebral edema and infarct volume. We postulated that glibenclamide administered during the early stages of stroke might foster neuroprotective microglial activity through ATP-sensitive potassium (KATP) channel blockade. We found in vitro that BV2 cell line showed upregulated expression of KATP channel subunits in response to pro-inflammatory signals and that glibenclamide increases the reactive morphology of microglia, phagocytic capacity and TNFα release. Moreover, glibenclamide administered to rats 6, 12 and 24h after transient Middle Cerebral Artery occlusion improved neurological outcome and preserved neurons in the lesioned core three days after reperfusion. Immunohistochemistry with specific markers to neuron, astroglia, microglia and lymphocytes showed that resident amoeboid microglia are the main cell population in that necrotic zone. These reactive microglial cells express SUR1, SUR2B and Kir6.2 proteins that assemble in functional KATP channels. These findings provide that evidence for the key role of KATP channels in the control of microglial reactivity are consistent with a microglial effect of glibenclamide into the ischemic brain and suggest a neuroprotective role of microglia in the early stages of stroke.
► Reactive microglia express KATP channels in tMCAo rat brain. ► Glibenclamide improves neurological outcome and neuroprotection in tMCAo rats. ► Microglial activation enhances KATP channel expression in vitro. ► KATP channel blockade modulates microglial reactivity and phagocytosis in vitro. ► Glibenclamide effects in stroke therapy involve microglia-mediated neuroprotection.
Senescence represents a stage in life associated with elevated incidence of morbidity and increased risk of mortality due to the accumulation of molecular alterations and tissue dysfunction, ...promoting a decrease in the organism's protective systems. Thus, aging presents molecular and biological hallmarks, which include chronic inflammation, epigenetic alterations, neuronal dysfunction, and worsening of physical status. In this context, we explored the AAV9‐mediated expression of the two main isoforms of the aging‐protective factor Klotho (KL) as a strategy to prevent these general age‐related features using the senescence‐accelerated mouse prone 8 (SAMP8) model. Both secreted and transmembrane KL isoforms improved cognitive performance, physical state parameters, and different molecular variables associated with aging. Epigenetic landscape was recovered for the analyzed global markers DNA methylation (5‐mC), hydroxymethylation (5‐hmC), and restoration occurred in the acetylation levels of H3 and H4. Gene expression of pro‐ and anti‐inflammatory mediators in central nervous system such as TNF‐α and IL‐10, respectively, had improved levels, which were comparable to the senescence‐accelerated‐mouse resistant 1 (SAMR1) healthy control. Additionally, this improvement in neuroinflammation was supported by changes in the histological markers Iba1, GFAP, and SA β‐gal. Furthermore, bone tissue structural variables, especially altered during senescence, recovered in SAMP8 mice to SAMR1 control values after treatment with both KL isoforms. This work presents evidence of the beneficial pleiotropic role of Klotho as an anti‐aging therapy as well as new specific functions of the KL isoforms for the epigenetic regulation and aged bone structure alteration in an aging mouse model.
Intraventricular administration of AAV vectors expressing secreted and transmembrane Klotho isoforms, rescued accelerated aging phenotype of SAMP8 mice. An improvement in cognitive and physical performance, recovery of epigenetic, inflammatory and senescence markers, as well as structural changes in long bones of these mice was detected.
Under pathological conditions, microglia, the resident CNS immune cells, become reactive and release pro-inflammatory cytokines and neurotoxic factors. We investigated whether this phenotypic switch ...includes changes in the expression of the L-type voltage-gated calcium channel (VGCC) in a rat model of N-methyl-d-aspartate-induced hippocampal neurodegeneration. Double immunohistochemistry and confocal microscopy evidenced that activated microglia express the L-type VGCC. We then analyzed whether BV2 microglia express functional L-type VGCC, and investigated the latter's role in microglial cytokine release and phagocytic capacity. Activated BV2 microglia express the CaV1.2 and CaV1.3 subunits of the L-type VGCC determined by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry. Depolarization with KCl induced a Ca2+ entry facilitated by Bay k8644 and partially blocked with nifedipine, which also reduced TNF-α and NO release by 40%. However, no nifedipine effect on BV2 microglia viability or phagocytic capacity was observed. Our results suggest that in CNS inflammatory processes, the L-type VGCC plays a specific role in the control of microglial secretory activity.
•Reactive microglial from hippocampus express α1 subunits of the L-type VGCC in vivo and in vitro.•Nifedipine and Bay k8644 modulate intracellular calcium concentration of reactive BV2 microglia.•L-type VGCC participates in the control of the pro-inflammatory activity of microglia.
Spontaneous neuronal activity is essential to neural development. Until recently, neurons were believed to be the only excitable cells to display spontaneous activity. However, cultured astrocytes ...and, more recently, astrocytes in situ are now known to exhibit spontaneous Ca2+ transients. Here we used Ca2+ imaging of astrocytes from transgenic mice for the simultaneous monitoring of Ca2+i changes in large numbers of astrocytes. We found that spontaneous activity is a common property of most brain astrocytes that is lost in response to a lesion. These spontaneous Ca2+i oscillations require extracellular and intracellular Ca2+. Moreover, network analysis revealed that most astrocytes formed correlated networks of dozens of these cells, which were synchronous with both astrocytes and neurons. We found that decreasing spontaneous Ca2+i transients in neurons by TTX does not alter the number of active astrocytes, although it impairs their synchronous network activity. Conversely, bicuculline-induced epileptic patterns of Ca2+i transients in neurons cause an increase in the number of active astrocytes and in their network synchrony. Furthermore, activation of non-NMDA and NMDA ionotropic glutamate receptors is required to correlate astrocytic networks. These results show that spontaneous activity in astrocytes and neurons is patterned into correlated neuronal/astrocytic networks in which neuronal activity regulates the network properties of astrocytes. This network activity may be essential for neural development and synaptic plasticity.
Background: In Hirschsprung’s disease (HSCR), a hypomorphic allele of a major gene, RET, accounts for most isolated (non-syndromic) cases, along with other autosomal susceptibility loci under a ...multiplicative model. However, some syndromic forms of HSCR are monogenic entities, for which the disease causing gene is known. Objective: To determine whether RET could be considered a modifier gene for the enteric phenotype on the background of a monogenic trait. Methods: The syndromic HSCR entities studied were congenital central hypoventilation (CCHS) and Mowat-Wilson syndrome (MWS), caused by PHOX2B and ZFHX1B gene mutations, respectively. The RET locus was genotyped in 143 CCHS patients, among whom 44 had HSCR, and in 30 MWS patients, among whom 20 had HSCR. The distribution of alleles, genotypes, and haplotypes was compared within the different groups. To test the interaction in vivo, heterozygous mice were bred for a null allele of Phox2b and Ret genes. Results:RET was shown to act as a modifier gene for the HSCR phenotype in patients with CCHS but not with MWS. The intestine of double heterozygote mice was indistinguishable from their littermates. A loss of over 50% of each gene function seemed necessary in the mouse model for an enteric phenotype to occur. Conclusions: In CCHS patients, the weak predisposing haplotype of the RET gene can be regarded as a quantitative trait, being a risk factor for the HSCR phenotype, while in MWS, for which the HSCR penetrance is high, the role of the RET predisposing haplotype is not significant. It seems likely that there are both RET dependent and RET independent HSCR cases.
Activation of mitochondrial ATP-sensitive potassium (K
ATP
) channels is postulated as an effective mechanism to confer cardio and neuroprotection, especially in situations associated to oxidative ...stress. Pharmacological activation of these channels inhibits glia-mediated neuroinflammation. In this way, diazoxide, an old-known mitochondrial K
ATP
channel opener, has been proposed as an effective and safe treatment for different neurodegenerative diseases, demonstrating efficacy in different animal models, including the experimental autoimmune encephalomyelitis (EAE), an animal model for Multiple Sclerosis. Although neuroprotection and modulation of glial reactivity could alone explain the positive effects of diazoxide administration in EAE mice, little is known of its effects on the immune system and the autoimmune reaction that triggers the EAE pathology. The aim of the present work was to study the effects of diazoxide in autoimmune key processes related with EAE, such as antigen presentation and lymphocyte activation and proliferation. Results show that, although diazoxide treatment inhibited in vitro and ex-vivo lymphocyte proliferation from whole splenocytes it had no effect in isolated CD4
+
T cells. In any case, treatment had no impact in lymphocyte activation. Diazoxide can also slightly decrease CD83, CD80, CD86 and major histocompatibility complex class II expression in cultured dendritic cells, demonstrating a possible role in modulating antigen presentation. Taken together, our results indicate that diazoxide treatment attenuates autoimmune encephalomyelitis pathology without immunosuppressive effect.
The aim of this study was to report the CT evolution and clinical significance of HCA after intra-arterial mechanical thrombectomy (revascularization by using retrievers and/or other mechanical ...devices without concomitant delivery of intra-arterial thrombolytics) in our patients. These lesions are common after intra-arterial thrombolysis, being considered a negative prognostic sign. Their significance after pure mechanical thrombectomy remains unknown.
Forty-eight patients were treated with mechanical thrombectomy by using retrievable stents between April 2010 and February 2011. All patients underwent initial (first 24 hours) and follow-up (48-72 hours) nonenhanced CT. We retrospectively analyzed the clinical and radiologic data of the patients with HCA and compared them with controls.
Fifteen of 48 patients presented with HCA. The site of occlusion was the MCA in 7 patients, both the extra- and intracranial segments of the ICA in 6, and the intracranial ICA in 2. In 7 patients, previous intravenous thrombolysis was administered. Complete recanalization (TICI 3) was achieved in 12 patients, and incomplete recanalization (TICI 2b), in 3. The location of HCA was the subarachnoid space in 6 patients, the brain parenchyma in 4 patients, and both in 5 patients. The HCA were asymptomatic in all patients. There was no statistical difference in final NIHSS score reduction (NIHSS pretreatment-NIHSS at discharge) between patients and controls.
In our series, HCA are common after mechanical thrombectomy but do not carry an increased risk of symptomatic hemorrhage or negative prognosis. These data might be related to the high rate of recanalization and the absence of intra-arterial thrombolytics.
Patterned intrinsic network activity plays a central role in shaping immature neuronal networks into functional circuits. However, the long-lasting signals that regulate spontaneous activity of ...developing circuits have not been identified. Here we study the net impact of TrkB signaling on early network activity of identified neuronal populations by analyzing postnatal hippocampi from trkB null mice. Ca2+ imaging showed that pyramidal neurons of trkB−/− mice displayed a decrease in intrinsic synchronous activity in neonatal animals but an increase in juveniles. Strikingly, alterations in network activity in trkB−/− hippocampus were associated with an aberrant induction of the transcription factor Fos. In contrast to pyramidal neurons, spontaneous Ca2+i oscillations in trkB−/− interneurons were consistently impaired throughout postnatal development. Moreover, the number of GABAergic synapses and the expression levels of GAD65 and KCC2 were decreased in mutant hippocampi, indicating that pre- and post-synaptic GABAergic components were impaired in trkB−/− mice. Finally, the partial blockade of GABAA receptor in postnatal slices revealed that mutant hippocampi displayed an increased susceptibility to network hyperexcitability. These results indicate that the lack of TrkB signaling during development impairs GABAergic neurotransmission, thereby leading to an age-dependent decrease followed by an increase in the intrinsic excitability of neuronal circuits. Furthermore, the present study indicates that long-lasting TrkB signaling may contribute to the construction of CNS circuits by modulating patterns of spontaneous Ca2+i oscillations.
Multiple Sclerosis (MS) is an acquired inflammatory demyelinating disorder of the central nervous system (CNS) and is the leading cause of nontraumatic disability among young adults. Activated ...microglial cells are important effectors of demyelination and neurodegeneration, by secreting cytokines and others neurotoxic agents. Previous studies have demonstrated that microglia expresses ATP-sensitive potassium (KATP) channels and its pharmacological activation can provide neuroprotective and anti-inflammatory effects. In this study, we have examined the effect of oral administration of KATP channel opener diazoxide on induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS.
Anti-inflammatory effects of diazoxide were studied on lipopolysaccharide (LPS) and interferon gamma (IFNγ)-activated microglial cells. EAE was induced in C57BL/6J mice by immunization with myelin oligodendrocyte glycoprotein peptide (MOG₃₅₋₅₅). Mice were orally treated daily with diazoxide or vehicle for 15 days from the day of EAE symptom onset. Treatment starting at the same time as immunization was also assayed. Clinical signs of EAE were monitored and histological studies were performed to analyze tissue damage, demyelination, glial reactivity, axonal loss, neuronal preservation and lymphocyte infiltration.
Diazoxide inhibited in vitro nitric oxide (NO), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) production and inducible nitric oxide synthase (iNOS) expression by activated microglia without affecting cyclooxygenase-2 (COX-2) expression and phagocytosis. Oral treatment of mice with diazoxide ameliorated EAE clinical signs but did not prevent disease. Histological analysis demonstrated that diazoxide elicited a significant reduction in myelin and axonal loss accompanied by a decrease in glial activation and neuronal damage. Diazoxide did not affect the number of infiltrating lymphocytes positive for CD3 and CD20 in the spinal cord.
Taken together, these results demonstrate novel actions of diazoxide as an anti-inflammatory agent, which might contribute to its beneficial effects on EAE through neuroprotection. Treatment with this widely used and well-tolerated drug may be a useful therapeutic intervention in ameliorating MS disease.
Acute vertebrobasilar occlusion is an ominous disease with few proved effective treatments. Experience with stent retrievers is scarce and limited to combined therapies (stent retrievers associated ...with previous intravenous fibrinolysis, intra-arterial thrombolysis, or other mechanical devices). We present our experience with 18 patients treated with direct thrombectomy by using stent retrievers.
Eighteen patients with vertebrobasilar occlusion were treated with direct thrombectomy by using stent retrievers at our hospital. The mean age was 67.5 years. Clinical presentation was sudden deterioration in consciousness level in 61.2% and progressive or fluctuating brain stem symptoms in 38.8%. Stroke subtype (TOAST) was atherothrombotic (33.3%), undetermined (33.3%), cardioembolic (27.7%), and of unusual etiology (5.5%).
The occlusion site was the vertebral artery in 1 case, proximal basilar artery in 4, middle basilar artery in 6, distal basilar artery in 5, and unilateral posterior cerebral artery in 2 cases. SRs included the Solitaire AB in 8 cases, Solitaire FR in 5 cases, and Trevo Pro in 5 cases. An 8F Merci balloon guide catheter was used in 15 patients, and a Neuron 6F, in 3 patients. Post-clot retrieval definitive intracranial stents were used in 5 patients (27.7%). Postprocedural TICI ≥ 2b was achieved in 17 patients (94.4%). Clinically, 72.2% of patients experienced an improved NIHSS score at discharge, 22.2% died, and in 5.5% the NIHSS scores did not change. The mRS score at 3 months was 0-2 in 9 patients (50%) and 3-5 in 5 patients (27.7%).
Thrombectomy with stent retrievers is feasible in the treatment of vertebrobasilar occlusion. These initial results must be confirmed by further prospective studies with a larger number of cases.
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